Development of a high throughput-compatible fluorescence-based assay for TRPM3 channel activity and surface expression.

Abstract

The Transient Receptor Potential Melastatin 3 (TRPM3) channel is a calcium-permeable non-selective cation channel that can be activated by heat and by a variety of chemical ligands, including neurosteroid pregnenolone sulfate (PS). The TRPM3 channel senses noxious heat in nociceptive neurons, and it is also expressed in the brain where its gating mechanisms and functions remain poorly understood. TRPM3 channel mutations in human patients were recently reported to be associated with intellectual disability and epilepsy, and to cause a gain of function phenotype by increasing the open probability of the channel. By using these mutations along with wild-type TRPM3 channels, expressed in tandem with a genetically encoded fluorescent calcium indicator, we developed and benchmarked a fluorescence-based assay to evaluate ion channel activity. We will apply this fluorescence-based assay to investigate genotype to phenotype relations in the TRPM3 channel at high-throughput. To distinguish between mutations affecting channel activity from those that impact surface expression in the plasma membrane, we are screening diverse engineered TRPM3 channel constructs that would enable specific fluorescence labelling of surface-expressed ion channels. This trafficking assay will further facilitate our effort to systematically identify residues in the transmembrane domain of the channel that are crucial for activation by different types of agonists.